Hepatitis C virus (HCV) is a leading cause of liver cancer worldwide. During infection, HCV induces the formation of rearranged membrane structures termed web. These membranes are also observed in vitro following expression of HCV NS4B protein alone. Various reports have shown that the web contains HCV nonstructural proteins as well as the viral genome, suggesting that this novel membrane might be the site where HCV genome replication occurs. This proposal will test the following hypotheses: (i) the web plays a critical role in HCV genome replication, (ii) the web contains several host factors, including Rab5, which play a direct or indirect role in HCV life cycle. To test these hypotheses, we propose the following Specific Aims: Aim I. Test whether the web plays a critical role in HCV genome replication. It is widely believed that the web provides the scaffold for HCV genome replication. However, there is no direct experimental evidence to prove this hypothesis. Our current assay for web formation consists of co-localization of punctate NS4B staining with endogenous Rab5B protein. This assay is generally confirmed by electron microscopy detection of the web. In this Aim, we will (IA) introduce mutation(s) in NS4B to abolish web formation and (IB) determine whether abolishing the web inhibits HCV genome replication. We will (1C) test whether NS4B oligomerization plays a role in web formation and genome replication. Aim II. Determine the significance of the presence of Rab5 protein in the web. Previous reports have shown that the web-inducing protein, NS4B, is associated mostly with the endoplasmic reticulum (ER) compartment. Our laboratory has recently found that early endosome markers, Rab5 and EEA1, are associated with web-inducing NS4B. In addition, Rab5 was found to interact with NS4B. In this Aim, we will (IIA) examine how Rab5 gets incorporated into the web. We will (IIB) identify the Rab5-associated proteins interacting with HCV NS4B. Finally, we will (IIC) determine whether the web alters endocytosis in the cell. Aim III. Determine the protein profile of the web. The association of web-inducing NS4B protein with ER and EE marker, Rab5, suggests that this novel membrane has a complex origin and composition. In our preliminary results, we have used lodixanol (Optiprep) gradient to isolate membrane fractions enriched in viral proteins involved in web formation and genome replication. In this Aim, we will (IIIA) combine Optiprep gradient with immunoprecipitation to isolate web-enriched membranes. We will (IIIB) use mass spectrometry to examine the protein profile of the immuno-isolated membrane. We hope that these studies will address the role of HCV-associated ultrastructural changes in the viral genome replication and viral persistence.